Aims: To assess the levels of hepcidin prohormone (Hep) in aqueous humor and plasma of human eyes with primary open-angle glaucoma (POAG) and to correlate their concentrations with the severity of glaucoma. Methods: Sixty patients with POAG and forty five patients with senile cataract (control group) were enrolled in the study prospectively. Aqueous humor samples were obtained by paracentesis from glaucoma and cataract patients who were undergoing elective surgery. Aqueous humor and corresponding plasma samples were analyzed for Hep concentrations by enzyme-linked immunosorbent assay. Results: Hep levels were significantly lower in aqueous humor of POAG patients with respect to the comparative group of cataract patients (P < 0.001). No significant difference in the levels of Hep in plasma of POAG and cataract patients. A positive correlation was found between plasma/aqueous humor Hep concentration in POAG versus the control group (P < 0.001). No significant correlation was found between Hep levels and the severity of visual field loss. Conclusion: Lower levels of aqueous humor Hep may be associated with POAG. In addition, Hep may be useful protein derivatives levels in aqueous humor of POAG patients as a consequence of glaucomatous damage.